DESCRIPTION (Adapted from the investigator's application): The long term goal of this project is to elucidate the relationship between the molecular structure and the functional behavior of voltage-dependent potassium channels. This will be accomplished by applying a new approach using electron paramagnetic resonance (EPR) spectroscopy to the study of the structure and dynamics of Shaker K+ Channels. K+ channels play a key role in a variety of cellular processes. They are directly involved in the generation and control of electrical potentials in nerve and muscle cells, the regulation of cell volume, the response of endocrine cells to their environment (insulin secretion) and the control of the heart rate. Consequently, efforts to understand K+ channel structure and function relate directly to human health and disease. High-resolution structural information in membrane proteins has been very difficult to obtain, mainly because of the limited sources of pure protein and the problems involved with their crystallization. The strategy we will pursue overcomes these difficulties. It is based on the replacement of native residues by cysteine using site-directed mutagenesis methods and subsequent labeling with a sulfhydryl-specific nitroxide spin label. Information on the structure and dynamics of the spin-labeled mutants can be obtained using EPR spectroscopy, which only requires a few micrograms of pure protein. To our advantage, the functional properties of each mutant can be studied by expression in Xenopus oocytes with conventional electrophysiological techniques. With these methods the investigators will 1) Obtain experimental information about the folding and topology of the Shaker K+ channel. 2) Obtain a set of intra-molecule and inter-molecule distances in order to constraint three-dimensional models of the channel. 3) Study voltage- dependent conformational changes correlated with channel activation and inactivation. Studies on the structure of K+ channels are both timely and relevant. Information on the structure of the super-family of voltage-dependent channels has been basically limited to their amino acid sequences and scattered information about their topology. New developments in EPR spectroscopy together with the availability of purified material in over-expressing systems will provide valuable information about the structural dynamics of these membrane proteins.